title: Quantification of dimethyl-ifosfamide and its N-deschloropropylated metabolites in mouse plasma by liquid chromatography-tandem mass spectrometry.
abstract: Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N(2)- and N(3)-deschloropropyl-dimethylifosfamide (N(2)-DCP-diMeIFO and N(3)-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289→168 for diMeIFO, m/z 213→168 for N(2)-DCP-diMeIFO, m/z 213→92 for N(3)-DCP-diMeIFO and m/z 261→154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20-10,000ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N(2)-DCP-diMeIFO, N(3)-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 μL plasma. The method was accurate with calculated bias from -5.8 to 4.0% for diMeIFO, from -1.1 to 10.6% for N(2)-DCP-diMeIFO and from -6.9 to 9.8% for N(3)-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice.
authors.affiliation: CNRS UMR 8203, Vectorologie et Thérapeutiques Anticancéreuses, Université Paris XI, Institut de Cancérologie Gustave Roussy, IRCIV, Villejuif, France. alain.deroussent@igr.fr
authors.forename: Alain
authors.initials: A
authors.lastname: Deroussent
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Rodriguez
authors.forename: Sonia
authors.initials: S
authors.lastname: Martelli
authors.forename: Atmane
authors.initials: A
authors.lastname: Seck
authors.forename: Estelle
authors.initials: E
authors.lastname: Dubus-Daudigeos
authors.forename: Didier
authors.initials: D
authors.lastname: Desmaële
authors.forename: Gilles
authors.initials: G
authors.lastname: Vassal
authors.forename: Angelo
authors.initials: A
authors.lastname: Paci
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D006801:Humans; D007069:Ifosfamide / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D016018:Least-Squares Analysis; D051379:Mice; D008819:Mice, Nude; D015203:Reproducibility of Results; D012208:Rhabdomyosarcoma / Q000097:blood / Q000378:metabolism; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007069:Ifosfamide